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WO2007124065A1 - Génération d'adn recombinant par clonage indépendant de séquence et indépendant de ligature - Google Patents

Génération d'adn recombinant par clonage indépendant de séquence et indépendant de ligature Download PDF

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Publication number
WO2007124065A1
WO2007124065A1 PCT/US2007/009679 US2007009679W WO2007124065A1 WO 2007124065 A1 WO2007124065 A1 WO 2007124065A1 US 2007009679 W US2007009679 W US 2007009679W WO 2007124065 A1 WO2007124065 A1 WO 2007124065A1
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sequence
vector
nucleotides
dna
length
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PCT/US2007/009679
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English (en)
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Stephen Elledge
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The Brigham And Women's Hospital, Inc.
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Priority to EP07775868A priority Critical patent/EP2079842A4/fr
Publication of WO2007124065A1 publication Critical patent/WO2007124065A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Definitions

  • the present invention is in the field of recombinant DNA technology and is directed to methodology for cloning DNA by homologous recombination, without the need for ligases.
  • MAGIC is an in vivo method that relies upon homologous recombination and bacterial mating (Li, et al, Nat. Gen. 37:311-319 (2005)).
  • Homologous recombination has important advantages over site-specific recombination in that it does not require specific sequences.
  • SLIC Sequence- and Ligation-lndependent Cloning
  • the invention is directed to a method of generating recombinant DNA by homologous recombination without the use of ligases. This is accomplished by amplifying one or more target DNA molecules by the polymerase chain reaction (PCR) using a forward primer and a reverse primer, each of which 15-100 (typically 15-50) nucleotides long.
  • the forward primer should terminate at one end in sequence A, wherein sequence A is 15-100 (typically 15-50) nucleotides in length, and the reverse primer should terminate at one end in a different sequence, sequence B, which is also 15-100 (typically 15- 50) nucleotides long.
  • a single stranded terminal region 15-100 nucleotides is generated in the amplified DNA molecules using exonuclease digestion so that, at one end, they have a 5' overhang corresponding, at least in part, to sequence A and, at the other end, a 5' overhang corresponding, at least in part, to sequence B.
  • These fragments are then annealed with a linearized vector that terminates at each end with a single stranded region 15-100 (typically 15-50) nucleotides in length.
  • One end should have a sequence, C, that is exactly complementary to sequence A, and the other end should have a sequence, D, that is exactly complementary to sequence B.
  • the final step in the process involves transforming a host cell (preferably E. coli) with the annealed complexes formed in step c). Enzymes in the host cell will then fill in any missing nucleotides and join the annealed DNA fragments together. If desired, this recombinant vector may now be recovered from the host. It should be noted that 3' overhangs will also work in this system.
  • a host cell preferably E. coli
  • the annealing of DNA fragments to vector may be done either in the presence of RecA (at a concentration of about 0.1-0.5 ng/ ⁇ l and preferably 0.2-0.4 ng/ ⁇ l) or in the absence of Rec A, but at a higher concentration (at least 0.5 ng/ ⁇ l and preferably 0.7-10.0 ng/ ⁇ l or higher).
  • the generation of single stranded regions in PCR amplified DNA and in the vector can be accomplished using one or more exonucleases such as: lambda nuclease; T7 nuclease; Exonuclease III; and T4 polymerase.
  • the invention is directed to method of generating recombinant DNA by homologous recombination without the use of ligases, in which single stranded regions are created by performing incomplete PCR.
  • This may be contrasted with ordinary PCR in that the extension step in the final cycle of denaturation, annealing and extension is omitted.
  • the method involves first amplifying one or more target DNA molecules in the manner described above but in which the final step in the PCR procedure does not include the extension of annealed DNA fragments with the Taq DNA polymerase, only the denaturation and renaturation, which results in incompletely extended DNA molecules annealing to produce dsDNA with 5' overhangs suitable for annealing. The amplified fragments are then annealed with vector and used to transform a host cell, again, as described above.
  • each fragment should be made to terminate at one end in a single stranded segment, either A or A', 15-100 nucleotides long ending in a 5' terminal phosphate and, at the other end, by a single stranded segment, B or B 1 , 15-100 nucleotides long also ending In a 5' terminal phosphate. It should be noted that 3' overhangs will also work in this embodiment.
  • Each A segment should consist of a sequence that is exactly complementary to at least one B sequence, with the exact sequences being chosen based upon the order in which the fragments should be arranged.
  • the DNA fragments produced should be subsequently, or concurrently, annealed to a linearized vector terminating at one end in a single stranded region 15-100 nucleotides in length and having a sequence C, that is exactly complementary to sequence A'.
  • the other end of the vector should also terminate in a single stranded region 15-100 nucleotides in length, but with a sequence, D, that is exactly complementary to sequence B 1 .
  • the final step is to transform a host cell, preferably E. coli, with the annealed complexes.
  • each A and A' segment has a sequence that is unique with respect to one another, i.e. there is a unique single stranded sequence associated with each fragment. This will promote the formation of a single arrangement of fragments annealed to the vector. Annealing reactions may be carried out either in the presence or absence of RecA, with the preferred host cell being E. coli.
  • the preferred method of generating single stranded end regions in fragment is through the use of an exonuclease such as: lambda nuclease; T7 nuclease; Exonuclease III; and T4 polymerase.
  • kits containing the various components needed to cany out the procedures described above may include at least one oligonucleotide 15-150 nucleotides in length that terminates at one end in sequence A, 15- 100 nucleotides in length and a vector that is, or can be, linearized to contain an end sequence that is 15-100 nucleotides long and that is exactly complementary to sequence A.
  • the kit should not include a DNA ligase but may include additional oligonucleotides (e.g., having a terminal sequence complementary to the other end of the vector) or additional components that may be used in the process, e.g., RecA or exonucleases.
  • Figure 1 In vitro recombination of MAGIC vectors mediated by RecA. A schematic for the production of recombinant DNA through in vitro homologous recombination and single-strand annealing.
  • FIG. 2 The dependency on RecA can be overcome by increased DNA concentrations.
  • One ⁇ g of linear vector pML385 and 1 ⁇ g of 40 bp homology Skpl insert fragment were treated with 0.5 U of T4 DNA polymerase for 1 hour.
  • the vector and inserts were then diluted and annealed with and without RecA in a 1:1 molar ratio at different concentrations.
  • Figure 3 Incomplete PCR (iPCR) and mixed PCR can be used to prepare inserts for SLIC cloning without nuclease treatment.
  • iPCR Incomplete PCR
  • P2F- P2R Primers PlF and P2R are longer than PlR and P2F and produce 5' and 3' overhangs respectively.
  • the two PCR products were mixed and heated to 95 0 C for 5 minutes to denature, and then cooled slowly to room temperature to reannealed.
  • FIG. 4 Multi-fragment assembly using SLIC.
  • A) A schematic illustrating the 3- way SLIC reaction with lacO oligos.
  • B) A schematic illustrating the 5-way SLIC reaction in which T4 DNA polymerase-treated linear vector, pML385, and inserts with different amounts of homology were annealed in equalmolar ratio and transformed.
  • the present invention is based upon studies demonstrating an efficient method for cloning that does not require the use of ligases.
  • the methodology can be applied to the cloning of a single known DNA sequence or to the transfer of a sequence from one vector to another.
  • PCR primers will be designed based upon known DNA sequences flanking the target sequence, i.e., the DNA to be cloned.
  • the methodology can be used to clone entire libraries using random DNA primers. The procedure is especially useful in the rapid assembly of numerous DNA fragments into a specific ordered arrangement within a vector.
  • nuclease digestion may be used to expose single stranded complementary or substantially complementary sequences on or near the ends of nucleic acid molecules and vectors.
  • the complementary or substantially complementary ends thus revealed are capable of being annealed.
  • Complementary nucleotides are A and T (or A and U), or C and G.
  • Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, and preferably 90%, 95%, or 100%.
  • "Completely" or "exactly” complementary sequences have no mismatches at all, i.e., all A's on one strand are aligned with Ts on the other, ail G's with Cs etc.
  • Hybridization refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide.
  • Hybridization conditions will typically include salt concentrations of less than about IM, more usually less than about 500 mM and less than about 200 mM.
  • Hybridization temperatures can be as low as 5°C, but are typically greater than 22°C, more typically greater than about 30 0 C, and preferably in excess of about 37°C.
  • Hybridizations are usually performed under stringent conditions, i.e. conditions under which a probe will hybridize to its target subsequence. Stringent conditions are sequence-dependent and are different in different circumstances.
  • stringent conditions are selected to be about 5 0 C, lower than the Tm for the specific sequence at s defined ionic strength and pH.
  • recombinant DNA is formed by annealing nucleic acid fragments to vectors with complementary or substantially complementary ends such that an overhang region is created from an end of the vector.
  • the overhang region may contain some sequences that are not complementary in addition to some that are complementary.
  • the nucleic acid fragment may be prepared and treated with nucleases to create a nucleic acid fragment with ssDNA ends that are complementary or substantially complementary to nuclease-treated ends of the vector.
  • the annealed complex may be transformed into a bacterial cell, such as E. coli.
  • nucleic acid fragments may exhibit increased efficiency in transformation in cells.
  • a nucleic acid fragment and a vector may be treated with a nuclease to create ssDNA ends that are capable of annealing such that an overhang region is excluded.
  • the nucleic acid fragment is treated with any exonuclease, such as lambda nuclease, T7 nuclease, Exonuclease III, and/or the exonuclease function of T4 polymerase.
  • Nucleic acid molecule assemblies may be created by annealing multiple nucleic acid fragments with each other and/or multiple vectors.
  • 3 -way, 5-way, or 10-way gene assemblies may be formed in which 3 nucleic acid molecules, 5 nucleic acid molecules, or 10 nucleic acid molecules are annealed together, respectively.
  • efficiency of annealing and/or transformation into cells > may be enhanced.
  • creation of the multiple gene assemblies may be increased up to 80-fold or greater upon preparation of inserts and addition of the fragments.
  • an overhang region may be created, the overhang region being of any sequence. Hence, there is no particular required sequence in the overhang region.
  • RecA protein enhances the efficiency of formation of annealed complexes of nucleic acid fragments and/or vectors such that small amounts of nucleic acid molecules and/or vectors may be stimulated for production of recombinant molecules by up to 100-fold or higher. Enhanced production of recombinant molecules may be achieved in the presence of RecA even at low concentrations of nucleic acid molecules.
  • Any given fragment may be directionally subcloned and used for high throughput subcloning of open reading frames (ORFs) into a given vector.
  • ORFs may be linked to different promoters or selectable markers.
  • site-directed mutagenesis of proteins may be accomplished in which any portion of a gene may be altered without the presence of restriction enzymes. Further, the gene may be reassembled with any sequence at any position. Also, fragments from one gene or coding sequence can be introduced into another gene, related gene, or coding sequence in frame.
  • Recombinant molecules can be assembled in vitro with any combination of fragments.
  • the fragments may include, for example, coding sequences, non-coding sequences, gene regulatory elements, whole genes, markers (e.g. nutritional, drug- resistance, enzymatic, colorimetric, fluorescent, etc.), origins of replication, recombination sites, retroviral components, etc.
  • a kit may be provided for generating designed recombinant nucleic acid constructs.
  • the kit may contain a nuclease.
  • Such a kit may additionally contain RecA.
  • the invention is concerned with nucleic acid molecules that contain a coding sequence for all or part of a human protein.
  • a plurality of nucleic acid molecules may be created for covering all or substantially all of the genes of the human genome.
  • the plurality of coding segments (including, without limitation, short coding sequences) may be provided on a microarray, substrate or plurality of substrates (e.g., microtiter wells, beads, microparticles and the like) and may each further encode a corresponding peptide or protein.
  • the encoded protein may further be utilized in protein or peptide display technologies.
  • each member of a plurality of short coding sequences may encode a corresponding peptide.
  • Each of the encoded and/or expressed peptides may overlap in its amino acid sequence with at least one other synthesized peptide such that all or substantially all, of the peptides encoded by the human genome are covered by the library.
  • Such coding sequences may encode antigenic peptide sequences, such as epitopes.
  • a biological sample from a subject may be brought into contact with a set of peptides containing linear epitopes.
  • the peptides may be provided in a display library in which immunoprecipitation procedures are carried out with the biological sample from the subject.
  • the biological sample may be, for example, patient serum or other bodily fluid from one or different individuals.
  • Such a sample also may be a tissue or cell sample, or a lysate or homogenate thereof.
  • a sample may be whole or fractionated; in some cases, a specific component (such as antibodies) may have been isolated from the sample for use in the methods of the invention.
  • the sample may contain antibodies, including for example, autoantibodies, which, upon exposure to and/or incubation with a display library of peptide epitopes of the human genome, may bind to the peptide epitopes.
  • the epitopes thus captured may be identified in any variety of ways. For example, corresponding coding sequences may be amplified using PCR from the co-affinity purified nucleic acid sequences. The molecules thus obtained may further be hybridized to coding regions on a microarray containing a plurality of coding regions of the human genome.
  • the coding sequences may be determined by such means without a pre- amplification step. Hence, a signature of auto-antibodies present in the patient sample may be determined.
  • “Microarray” refers to a type of multiplex assay product that comprises a solid phase support having a substantially planar surface on which there is an array of spatially defined non-overlapping regions or sites that each contain an immobilized hybridization probe.
  • “Substantially planar” means that features or objects of interest, such as probe sites, on a surface may occupy a volume that extends above or below a surface and whose dimensions are small relative to the dimensions of the surface. For example, beads disposed on the face of a fiber optic bundle create a substantially planar surface of probe sites, or oligonucleotides disposed or synthesized on a porous planar substrate creates a substantially planar surface. Spatially defined sites may additionally be "addressable" in that its location and the identity of the immobilized probe at that location are known or determinable.
  • the oligonucleotides or polynucleotides on microarrays are single stranded and are covalently attached to the solid phase support, usually by a 5'-end or a 3'- end.
  • the density of non-overlapping regions containing nucleic acids in a microarray is typically greater than 100 per cm 2 , and more preferably, greater than 1000 per cm 2 .
  • Microarray technology relating to nucleic acid probes is reviewed in the following exemplary references: Schena, editor, Microarravs: A Practical Approach (IRL Press, Oxford, 2000); Southern, Current Opin. Chem. Biol. 2:404-410 (1998); Nature Genetics Supplement, 27:1-60 (1999); and Fodor et al, U.S. Pat.
  • Microarrays may be formed in a variety of ways, as disclosed in the following exemplary references: Brenner, et al, Nature Biotechnology 75:630-634 (2000); US Pat. No. 6,133,043; US Pat. No. 6,396,995; US Pat. No. 6,544,732; and the like.
  • “Microarrays” or “arrays” can also refer to a heterogeneous pool of nucleic acid molecules that is distributed over a support matrix.
  • the nucleic acids can be covalently or noncovalently attached to the support.
  • the nucleic acid molecules are spaced at a distance from one another sufficient to permit the identification of discrete features of the array.
  • Nucleic acids on the array may be non-overlapping or partially overlapping.
  • “Amplifying” includes the production of copies of a nucleic acid molecule of the array or a nucleic acid molecule bound to a bead via repeated rounds of primed enzymatic synthesis.
  • “In situ” amplification indicates that the amplification takes place with the template nucleic acid molecule positioned on a support or a bead, rather than in solution. In situ amplification methods are described in US 6,432,360.
  • “Support” can refer to a matrix upon which nucleic acid molecules of a nucleic acid array are placed.
  • the support can be solid or semi-solid or a gel.
  • “Semi-solid” refers to a compressible matrix with both a solid and a liquid component, wherein the liquid occupies pores, spaces or other interstices between the solid matrix elements.
  • Semi-solid supports can be selected from polyacrylamide, cellulose, polyamide (nylon) and crossed linked agarose, dextran and polyethylene glycol.
  • Randomly-patterned or random refers to non-ordered, non-Cartesian distribution (in other words, not arranged at pre-determined points along the x- or y- axes of a grid or at defined “clock positions,” degrees or radii from the center of a radial pattern) of nucleic acid molecules over a support, that is not achieved through an intentional design (or program by which such design may be achieved) or by placement of individual nucleic acid features.
  • Such a "randomly-patterned" or “random” array of nucleic acids may be achieved by dropping, spraying, plating or spreading a solution, emulsion, aerosol, vapor or dry preparation comprising a pool of nucleic acid molecules onto a support and allowing the nucleic acid molecules to settle onto the support without intervention in any manner to direct them to specific sites thereon.
  • Arrays of the invention can be randomly patterned or random.
  • Heterogeneous refers to a population or collection of nucleic acid molecules that comprises a plurality of different sequences. According to one aspect, a heterogeneous pool of nucleic acid molecules results from a preparation of RNA or DNA from a cell which may be unfractionated or partially-fractionated.
  • Oligonucleotide or “polynucleotide,” which are used synonymously, means a linear polymer of natural or modified nucleosidic monomers linked by phosphodiester bonds or analogs thereof.
  • oligonucleotide usually refers to a shorter polymer, e.g. comprising from about 3 to about 100 monomers, and the term “polynucleotide” usually refers to longer polymers, e.g. comprising from about 100 monomers to many thousands of monomers, e.g. 10,000 monomers, or more.
  • Oligonucleotides comprising probes or primers usually have lengths in the range of from 12 to 100 nucleotides, and more usually, from 18 to 40 nucleotides.
  • Oligonucleotides and polynucleotides may be natural or synthetic. Unless otherwise indicated, whenever an oligonucleotide is represented by a sequence of letters, such as "ATGC,” it will be understood that the nucleotides are in 5 1 to 3 1 order from left to right and that "A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, "T” denotes deoxythymidine, and “U” denotes the ribonucleoside, uridine. Usually oligonucleotides comprise the four natural deoxynucleotides; however, they may also comprise ribonucleosides or non-natural nucleotide analogs.
  • Oligonucleotide tag or “tag” means an oligonucleotide that is attached to a polynucleotide and is used to identify and/or track the polynucleotide in a reaction.
  • a oligonucleotide tag is attached to the 3'- or 5'-end of a polynucleotide to form a linear conjugate, sometime referred to herein as a "tagged polynucleotide,” or equivalently, an "oligonucleotide tag-polynucleotide conjugate,” or “tag-polynucleotide conjugate.”
  • tagged polynucleotide or equivalently, an "oligonucleotide tag-polynucleotide conjugate," or “tag-polynucleotide conjugate.”
  • oligonucleotide tags can each have a length within a range of from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides, respectively.
  • a tag that is useful in the present invention to identify samples captured from a specific patient or other source is of sufficient length and complexity to distinguish it from sequences that identify other patients or sources of DNA being assayed in parallel.
  • identification of the captured epitopes and antibodies may be accomplished in any variety of ways.
  • captured phage encoding epitopes of interest can be sequenced or identified by hybridization to microarrays. Once identified, particular epitopes might be identified in any of a number of additional ways as peptide reagents.
  • labeled epitopes are detected via a microarray of tag epitopes.
  • tag epitopes For each different antibody (e.g., from distinct patients, patient samples or other sources) there is a unique labeled epitope tag. That is, the pair consisting of (i) the sequence of the epitope tag and (ii) a label that generates detectable signal are uniquely associated with a particular locus.
  • the nature of the label on an epitope tag can be based on a wide variety of physical or chemical properties including, but not limited to, light absorption, fluorescence, chemiluminescence, electrochemi-luminescence, mass, charge, and the like.
  • the signals based on such properties can be generated directly or indirectly.
  • a label can be a fluorescent molecule covalently attached to an epitope tag that directly generates an optical signal.
  • a label can comprise multiple components, such as a hapten-antibody complex, that, in turn, may include fluorescent dyes that generated optical signals, enzymes that generate products that produce optical signals, or the like.
  • the label on a tag is a fluorescent label that is directly or indirectly attached to a tag.
  • such fluorescent label is a fluorescent dye or quantum dot selected from a group consisting of from 2 to 6 spectrally resolvable fluorescent dyes or quantum dots.
  • one or more fluorescent dyes are used as labels for labeled target sequences, e.g. as disclosed by US Pat. No. 5,188,934 (4,7-dichlorofluorscein dyes); US Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes); US Pat. No. 5,847,162 (4,7- dichlororhodamine dyes); US Pat. No. 4,318,846 (ether-substituted fluorescein dyes); US Pat. No. 5,800,996 (energy transfer dyes); US Pat. No. 5,066,580 (xanthene dyes): US Pat. No. 5,688,648 (energy transfer dyes); and the like.
  • Labeling can also be carried out with quantum dots, as disclosed in the following patents and patent publications: US Pat. Nos. 6,322,901; 6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392; 2002/0045045; 2003/0017264; and the like.
  • fluorescent nucleotide analogues readily incorporated into the labeling oligonucleotides include, for example, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ., USA), fluorescein- 12-dUTP, tetramethylrhodamine-6-dUTP, Texas RedTM.-5-dUTP, Cascade BlueTM.-7-dUTP, BODIPYTM. FL-14-dUTP, BODIPYTM.R- 14-dUTP, BODIPYTM. TR-14-dUTP, Rhodamine GreenTM.-5-dUTP, Oregon GreenRTM.
  • TMR-14-UTP BODIPYTM. TR-14-UTP, Rhodamine GreenTM.-5-UTP, Alexa FluorTM. 488-5-UTP, Alexa FluorTM. 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg., USA).
  • Biotin may also be used as a label on a detection oligonucleotide, and subsequently bound by a detectably labeled avidin/streptavidin derivative (e.g. phycoerythrin-conjugated streptavidin), or a detectably labeled anti-biotin antibody.
  • Digoxigenin may be incorporated as a label and subsequently bound by a detectably labeled anti-digoxigenin antibody (e.g. fluoresceinated anti-digoxigenin).
  • an aminoallyl-dUTP residue may be incorporated into a detection oligonucleotide and subsequently coupled to an N-hydroxy succinimide (NHS) derivitized fluorescent dye, such as those listed supra.
  • NHS N-hydroxy succinimide
  • any member of a conjugate pair may be incorporated into a detection oligonucleotide provided that a detectably labeled conjugate partner can be bound to permit detection.
  • the term antibody refers to an antibody molecule of any class, or any subfragment thereof, such as an Fab.
  • PCR Polymerase chain reaction
  • PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates.
  • the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument.
  • a double stranded target nucleic acid may be denatured at a temperature >90°C, primers annealed at a temperature in the range 50-75 0 C, and primers extended at a temperature in the range 72-78°C.
  • PCR encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like. Reaction volumes range from a few hundred nanoliters, e.g. 200 nL, to a few hundred microliters, e.g., 200 microliters.
  • Reverse transcription PCR or "RT-PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified.
  • Auto-antibodies may be detected and characterized in a patient's sera or other sample. Auto-antibodies may be present in patient sera in any number of conditions including, but not limited to, scleroderma, arthritis, multiple sclerosis, lupus, etc. A biological sample, such as patient serum, may be obtained from the patient and exposed or incubated with epitopes as described. The auto-antibodies may be identified to determine a signature of auto-antibodies in the sera. Hence, more effective and rapid diagnosis may be accomplished for the patent with subsequent directed therapy.
  • auto-immune responses may be identified in various cancers.
  • a slow growing tumor may be undetectable in the early stages of the disease.
  • identification of autoantibodies in a patient sample may provide clues or early diagnosis of the development of the cancer even before the cancer is advanced enough to diagnose using alternative diagnostic modalities.
  • more effective and earlier diagnosis of cancer may be accomplished by identifying the presence of autoantibodies in a patient's sample.
  • the peptides may also be used to search for proteins other than antibodies.
  • a linear protein or peptide segment may bind to a given target protein in vitro.
  • the linear protein or peptide segment may be detected in a sample via binding to a target protein.
  • the target protein may contain an epitope encoded in the human genome.
  • the process may further be used to identify proteins.
  • the present example describes a novel cloning method SLIC (Sequence and Ligation-Independent Cloning) that allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing.
  • SLIC mimics in vivo homologous recombination by relying on exonuclease generation of single strand DNA (ssDNA) overhangs on insert and vector fragments and the assembly of these fragments by recombination in vitro.
  • SLIC inserts can be prepared by incomplete PCR (/PCR) or mixed PCR.
  • SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously.
  • SLIC circumvents the sequence requirements of traditional methods and is much more sensitive when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.
  • pML403 To remove the lacO site from pML385, we created pML403 by annealing a pair of oligonucleotides, MZL571 and MZL572, and cloning them into Notl-Sacl cleaved pML385.
  • plasmid tmGIPZ-pheS by inserting the PheS Gly294 gene, amplified by PCR using primers MZL590 and MZL591, into the Mlul-Xhol cleaved tmGIPZ.
  • Inserts are amplified using Taq DNA polymerase.
  • a 100 ⁇ l PCR reaction was set up with 250 ⁇ M of each dNTP, 0.5 ⁇ M of each primer, and 2.5 U of Taq DNA polymerase (from Eppendorf). Cycle as following: 94 0 C for 45 seconds; 30 cycles of 94°C for 45 seconds, 54°C for 45 seconds, and 72°C for 1 minute; 72°C for 10 minutes.
  • Add 20 U ofDpnl to 100 ⁇ l of PCR products after PCR, incubate at 37°C for 1 hour (not necessary if going from a MAGIC vector to CoIEl origin).
  • the PCR products are purified by QIAquick PCR purification column.
  • Inserts are amplified using Taq DNA polymerase.
  • a 100 ⁇ l PCR reaction was set up with 250 ⁇ M of each dNTP, 0.5 ⁇ M of each primer, and 2.5 U of Taq DNA IS polymerase (from Eppendorf). Cycle as following: 94°C for 45 seconds; 30 cycles of 94°C for 45 seconds, 54°C for 45 seconds, and 72°C for 1 minute; 72°C for 10 minutes.
  • Add 20 U of Dpnl to 100 ⁇ l of PCR products after PCR, incubate at 37°C for 1 hour.
  • the PCR products were purified by QIAquick PCR purification column. Quantitate PCR products.
  • iPCR insert the PCR product is heated to 95°C for 5 minutes to denature, cooled slowly to room temperature in 1 hour to renature, dilute and proceed to annealing reaction.
  • mixed PCR inserts the two PCR products are mixed in equal amounts and heated to 95°C for 5 minutes to denature, cooled slowly to room temperature for 1 hour to renature, dilute and proceed to annealing reaction.
  • SLIC with iPCR or mixed PCR products iPCR products were generated under the same conditions as regular PCR and purified by a QIAquick PCR purification column. We denatured the purified iPCR product at 95 0 C and renatured slowly to room temperature in one hour. We annealed the vector and inserts at a 1:1 molar ratio and transformed.
  • Homologous recombination in vivo depends upon a double-stranded break, generation of ssDNA by exonucleases, homology searching by recombinases, annealing of homologous stretches and repair of overhangs and gaps by enzymes that include resolvases, nucleases and polymerases.
  • enzymes that include resolvases, nucleases and polymerases We reasoned it might be possible to generate recombination intermediates in vitro and introduce these into cells to allow the cells endogenous repair machinery to finish the repair to generate recombinant DNA ( Figure 1).
  • exonucleases to chew back one strand to reveal ssDNA overhangs.
  • T4 DNA polymerase which produces 5' overhangs, because it gave the best and most reproducible results and had the ability to terminate excision by addition of a single dNTP.
  • T4 DNA polymerase was generated the vector by cleavage with a restriction enzyme and insert was generated by PCR.
  • T4 DNA polymerase was treated with T4 DNA polymerase in the absence of dNTPs to generate overhangs, then incubated vector and insert with and without RecA protein and ATP to promote recombination, and transformed into E. coli. Vector alone gave some background we traced to a small amount of uncleaved vector.
  • MAGIC donor vectors have greater than 60 bp homology with recipient vectors on each end and generate inserts by cleavage with I-Scel of both donor and recipient plasmids.
  • the recipient used is a Lenti vector and the donor fragment is an shRNA cassette from an shRNA library.
  • vector alone gave 8 transformants per ng of vector whereas vector plus insert gave 3,900, a 500-fold stimulation of recombination, similar to what is seen in vivo.
  • Ten of ten clones yielded restriction fragments consistent with the predicted restriction map.
  • the isolation of 3,900 transformants per ng of vector means libraries can be transferred by this method in vitro without losing complexity.
  • recombinant DNA assembled by SLIC achieves a seamless transfer of genetic elements in vitro without the need for specific sequences required for ligation or site-specific recombination. This is accomplished by harnessing the power of homologous recombination in vitro to assemble recombinant DNA that resemble recombination intermediates such as gapped or branched molecules which upon introduction into bacteria are repaired to regenerate a double-stranded, covalently closed plasmids.
  • recombinant DNA can be assembled efficiently with very small amounts of DNA.
  • Homologous recombination events that occur in vivo such as those carried out by MAGIC cloning can be efficiently recapitulated in vitro using SLIC.
  • This method can be used to assemble DNA made by PCR or restriction fragments. The only requirement is that the fragments to be assembled contain on their ends sequences of 20 bp or longer to allow stable annealing. Excision by the proofreading exonuclease of T4 DNA polymerase has proven to be the most reproducible and easiest to manipulate method for generating 5' overhangs. Although much less efficient, iPCR also gives substantial stimulation of transformation. This might be sufficient for routine subcloning purposes although there is likely to be more variable depending on the completeness of the PCR synthesis.
  • PCR primers for inserts are designed to contain appropriate 5' extension sequences lacking a particular dNTP that, after treatment with T4 DNA polymerase in the presence of the particular dNTP, generates specific 12 nucleotide ssDNA overhangs that are complementary to overhangs engineered into the vector.
  • these overhangs have sequence constraints as they must be devoid of a common dNTP, which limits their use to specialized vectors bearing that sequence.
  • the realization that alternative recombination intermediates with imprecise junctions such as large gaps and overhangs can be efficiently repaired in vivo completely liberates SLIC from the sequence constraints that the LIC method suffers. Having the ability to generate overlaps of greater lengths of unrestrained sequence provides much greater utility for SLIC and its combination with RecA makes it able to function at much lower DNA concentrations.
  • SLIC Slica-resistant plasmids
  • SLIC allows alterations of fragments internal to a gene borne on a plasmid. For example it would be simple to introduce a PCR fragment into a restriction site in vitro even if that fragment contained multiple sites for the enzyme. Also, since the homologous junctions of fragments can be controlled, SLIC offers a new approach to the generation of site-directed mutations.
  • SLIC works with PCR fragments while multi-site Gateway has only been demonstrated with cloned fragments on donor plasmids.
  • the ability to assemble complex combinations of DNA sequence elements in defined orders will be particularly important in the field of synthetic biology. No attempts were made to optimize the 10-way assemblies and it is likely one could significantly improve the yield in future experiments. Thus, it is likely that molecules with greater than 10 fragments will be able to be assembled in the future.
  • the utility of the SLIC system is not limited to gene assembly. Genetic elements of any kind can be assembled using this system.
  • vectors being assembled in a combinatorial fashion from component parts. For example, using the highly efficient 5-way assembly one could combine an open reading frame together with a particular epitope tag, a tissue specific promoter, a retroviral vector together with a selectable marker of choice to generate a custom expression assembly.
  • a selectable marker of choice to generate a custom expression assembly.
  • vectors might exist in virtual form and be assembled in final form as needed.
  • SLIC now brings the ability to manipulate DNA sequences with much greater facility than previously possible.
  • Other complex assemblies such as homologous recombination targeting vectors could be assembled in one step by SLIC.
  • the linear vector2 (ptmGIPZ-pheS, 12 kb) and insert (using primer pair P1F-P2R) were treated with T4 DNA polymerase.
  • the vector and the insert generated by T4 DNA polymerase were annealed at 1:2 molar ratio of vector to insert and transformed.
  • the vector and the insert generated by mixed PCR were annealed at 1:6 molar ratio of vector to insert and transformed.
  • Table 5 Three-way SLIC with lacO selection.
  • Table 6 Five-wa SLIC with different amounts of homolo .

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Abstract

La présente invention concerne des procédés de clonage d'ADN par recombinaison homologue. Les procédés peuvent être utilisés sans nécessiter de ligases ou d'enzymes de restriction et permettent l'alignement rapide d'une pluralité de fragments d'ADN.
PCT/US2007/009679 2006-04-21 2007-04-20 Génération d'adn recombinant par clonage indépendant de séquence et indépendant de ligature WO2007124065A1 (fr)

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7435562B2 (en) 2000-07-21 2008-10-14 Modular Genetics, Inc. Modular vector systems
US7485423B2 (en) 2004-11-11 2009-02-03 Modular Genetics, Inc. Ladder assembly and system for generating diversity
US7579146B2 (en) 1999-01-05 2009-08-25 Trustees Of Boston University Nucleic acid cloning
EP2175018A1 (fr) * 2008-10-08 2010-04-14 Icon Genetics GmbH Procédé de clonage précis
US7723077B2 (en) 2005-08-11 2010-05-25 Synthetic Genomics, Inc. In vitro recombination method
US7776532B2 (en) 2005-08-11 2010-08-17 Synthetic Genomics, Inc. Method for in vitro recombination
US8129119B2 (en) 1999-01-05 2012-03-06 Modular Genetics, Inc. System for manipulating nucleic acids
CN103160495A (zh) * 2011-12-15 2013-06-19 张力军 一种非连接依赖性的快速克隆方法
CN104630178A (zh) * 2015-01-28 2015-05-20 苏州神洲基因有限公司 一种混合蛋白酶体系、包含该混合蛋白酶体系的试剂盒及其使用方法
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AU2017239633B2 (en) * 2010-08-16 2019-07-18 Salk Institute For Biological Studies Adenoviral assembly method
US20200224207A1 (en) * 2017-07-05 2020-07-16 OriCiro Genomics, Inc. Dna production method and dna fragment-joining kit
WO2021064115A1 (fr) 2019-10-01 2021-04-08 Universitetet I Tromsø - Norges Arktiske Universitet Procédé et kit d'assemblage de multiples fragments d'adn à température ambiante
WO2021064106A1 (fr) 2019-10-01 2021-04-08 Universitetet I Tromsø - Norges Arktiske Universitet Adn polymérase et exonucléase 3' à 5' dérivée de l'adn polymérase
US11077156B2 (en) 2013-03-14 2021-08-03 Salk Institute For Biological Studies Oncolytic adenovirus compositions
US11130968B2 (en) 2016-02-23 2021-09-28 Salk Institute For Biological Studies High throughput assay for measuring adenovirus replication kinetics
US11401529B2 (en) 2016-02-23 2022-08-02 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
WO2023147547A1 (fr) * 2022-01-31 2023-08-03 Integrated Dna Technologies, Inc. Procédés et compositions d'assemblage d'adn basé sur la recombinaison
US11813337B2 (en) 2016-12-12 2023-11-14 Salk Institute For Biological Studies Tumor-targeting synthetic adenoviruses and uses thereof

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030017552A1 (en) * 2000-07-21 2003-01-23 Jarrell Kevin A. Modular vector systems
US20080014616A1 (en) * 2006-07-11 2008-01-17 Kevin Jarrell Methods of introducing targeted diversity into nucleic acid molecules
WO2010045326A1 (fr) * 2008-10-14 2010-04-22 Barnes Wayne M Procédés de recombinaison in vitro utilisant une exonucléase 5’
US8623652B2 (en) * 2009-04-06 2014-01-07 Lucigen Corporation Host-vector system for cloning and expressing genes
US20120122158A1 (en) * 2010-05-18 2012-05-17 Bio-Rad Laboratories, Inc. Pcr for dna that is resistant to amplification
US9689012B2 (en) 2010-10-12 2017-06-27 Cornell University Method of dual-adapter recombination for efficient concatenation of multiple DNA fragments in shuffled or specified arrangements
US20120295311A1 (en) * 2011-05-09 2012-11-22 Basf Se Methods and Materials for Nucleic Acid Manipulation
WO2014153188A2 (fr) 2013-03-14 2014-09-25 Life Technologies Corporation Synthèse hautement efficace de petits volumes d'acides nucléiques
EP2768607B1 (fr) 2011-09-26 2021-08-18 Thermo Fisher Scientific GENEART GmbH Plaque multipuits pour la synthèse hautement efficace de petits volumes d'acides nucléiques
CA2906556C (fr) 2013-03-15 2022-07-05 Gen9, Inc. Compositions et procedes pour la synthese d'acides nucleiques multiplexes
WO2016094512A1 (fr) 2014-12-09 2016-06-16 yLIFE TECHNOLOGIES CORPORATION Synthèse hautement efficace de petits volumes d'acides nucléiques
EP3763818A1 (fr) 2015-10-06 2021-01-13 Pierce Biotechnology, Inc. Dispositifs et procédés de production de protéines
WO2018013250A1 (fr) * 2016-07-14 2018-01-18 Gene Infinity Llc Procédés de clonage rapide indépendant de la ligature d'adn et leurs utilisations
US20210147830A1 (en) 2018-06-29 2021-05-20 Thermo Fisher Scientific Geneart Gmbh High throughput assembly of nucleic acid molecules
GB201905303D0 (en) 2019-04-15 2019-05-29 Thermo Fisher Scient Geneart Gmbh Multiplex assembly of nucleic acid molecules
WO2021178809A1 (fr) 2020-03-06 2021-09-10 Life Technologies Corporation Synthèse et assemblage d'acide nucléique à fidélité de séquence élevée
CN113215145B (zh) * 2021-05-08 2022-07-08 河南大学 一种不依赖pcr反应的短片段克隆方法
WO2024132094A1 (fr) 2022-12-19 2024-06-27 Thermo Fisher Scientific Geneart Gmbh Extraction de molécules d'acide nucléique à séquence vérifiée
WO2024155610A1 (fr) * 2023-01-18 2024-07-25 Zhenyu Zhu Procédure de clonage sans soudure à période de récupération statique
GB2628173A (en) 2023-03-17 2024-09-18 Thermo Fisher Scient Geneart Gmbh Methods of producing modified nucleic acid sequences for eliminating adverse splicing events

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL120338A0 (en) * 1997-02-27 1997-06-10 Gesher Israel Advanced Biotecs Single step DNA fragments assembly
US7575860B2 (en) * 2000-03-07 2009-08-18 Evans David H DNA joining method
US7435562B2 (en) * 2000-07-21 2008-10-14 Modular Genetics, Inc. Modular vector systems

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAUN ET AL.: "Rapid, Reliable Ligation-Independent Cloning of PCR Products Using Modified Plasmid", BIOTECHNIQUES, vol. 13, no. 4, 1992, pages 514 - 517, XP008103670 *

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US7485423B2 (en) 2004-11-11 2009-02-03 Modular Genetics, Inc. Ladder assembly and system for generating diversity
US11542529B2 (en) 2005-08-11 2023-01-03 Codex Dna, Inc. In vitro recombination method
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US20200224207A1 (en) * 2017-07-05 2020-07-16 OriCiro Genomics, Inc. Dna production method and dna fragment-joining kit
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WO2023147547A1 (fr) * 2022-01-31 2023-08-03 Integrated Dna Technologies, Inc. Procédés et compositions d'assemblage d'adn basé sur la recombinaison

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